Inhibitors of janus kinases

ABSTRACT

The instant invention provides for compounds that inhibit the four known mammalian JAK kinases (JAK1, JAK2, JAK3 and TYK2). The invention also provides for compositions comprising such inhibitory compounds and methods of inhibiting the activity of JAK1, JAK2, JAK3 and TYK2 by administering the compound to a patient in need of treatment for myeloproliferative disorders or cancer.

BACKGROUND OF THE INVENTION

Janus kinase (JAK) is a family of intracellular non-receptor tyrosinekinases, ranging from 120-140 kDa, that transduce cytokine-mediatedsignals via the JAK-STAT pathway. The JAK family plays a role in thecytokine-dependent regulation of proliferation and function of cellsinvolved in immune response. Currently, there are four known mammalianJAK family members: JAK1, JAK2, JAK3 and TYK2.

JAK1, JAK2 and TYK2 are ubiquitously expressed whereas JAK3 is expressedin the myeloid and lymphoid lineages. The JAK family members arenon-receptor tyrosine kinases that associate with many hematopoietincytokines, receptor tyrosine kinases and GPCR's. JAK1−/− mice were foundto be developmentally similar to the JAK1+/+ although they weighed 40%less than the wild-type and failed to nurse at birth. These pups werenot viable and died within 24 hours of birth (Meraz et al Cell, 1998,373-383). JAK1 deficiency led to reduced number of thymocytes, pre-Bcells and mature T and B lymphocytes. TYK2(−/−) mice, on the other hand,are viable, demonstrating subtle defects in their response to IFN-α/βand IL-10 and profound defects to the response of IL-12 and LPS.

The breast cancer susceptibility protein (BRCA1) acts as a tumorsuppressor and contributes to cell proliferation, cycle regulation, aswell as DNA damage and repair. BRCA1 (−/−) mice develop normally but dieby 7.5 days post embryo suggesting a key role of BRCA1 for development.Mice in which the BRCA1 protein was overexpressed led to inhibition ofcell growth and sensitized cells to cytotoxic reagents. In the humanprostate cancer cell line Du-145 (Gao FEES Letters 2001, 488, 179-184),enhanced expression of BRCA1 was found to correlate with constitutiveactivation of STAT3 as well as activation of JAK1 and JAK2. Moreover,antisense oligonucleotides selective for STAT3 led to significantinhibition of cell proliferation and apoptosis in Du-145 cells. Thisdata supports the potential utility of JAK1 and JAK2 inhibitors in thetreatment of prostate cancer.

Campbell et al (Journal of Biological Chemistry 1997, 272, 2591-2594) asreported that STAT3 is constitutively activated v-Src transformed cells.To test whether STAT3 activation resulted via signaling through theJAK-STAT pathway, three fibroblast cell lines (NIH3T3, Balb/c, and 3Y1)were transformed with v-Src. The level of JAK1 phosphorylation in NIH3T3cells was markedly increased in cells overexpressed with v-Src or mutantc-Src (Y527F) compared to those in the less transforming c-Src. Thisresult correlated with increased JAK1 enzymatic activity. Similarresults were observed with JAK2 albeit to a lesser extent. These resultsare consistent with constitutive activation of JAK1 and possibly JAK2which contribute to the hyperactivation of STAT3 in Src-transformedcells.

Asthma is a disease that is increasing in prevalence and results in“airway obstruction, airway hyperresponsiveness, and airway inflammationand remodeling” (Pernis The Journal of Clinical Investigation 2002, 109,1279-1283). A common cause is the inappropriate immune responses toenvironmental antigens usually involving CD4+ T helper cells (TH2) whichare triggered from cytokines IL-4, IL-5, IL-6, IL-10, and IL-13 whichsignal through JAK1/JAK3-STAT6 pathway. Th1 cells are thought to beinvolved with the “delayed-type hypersensitivity responses” whichsecrete IL-2, IFN-γ, and TNF-β and signal through the JAK2/TYK2-STAT4pathway. STAT6 (−/−) mice were protected from AHR when challenged withenvironmental antigens and showed no increase in IgE levels or thequantity of mucous containing cells.

JAK2 is a cytoplasmic protein-tyrosine kinase that catalyzes thetransfer of the gamma-phosphate group of adenosine triphosphate to thehydroxyl groups of specific tyrosine residues in signal transductionmolecules. JAK2 mediates signaling downstream of cytokine receptorsafter ligand-induced autophosphorylation of both receptor and enzyme.The main downstream effectors of JAK2 are a family of transcriptionfactors known as signal transducers and activators of transcription(STAT) proteins. Studies have disclosed an association between anactivating JAK2 mutation (JAK2V617F) and myeloproliferative disorders.The myeloproliferative disorders, a subgroup of myeloid malignancies,are clonal stem cell diseases characterized by an expansion ofmorphologically mature granulocyte, erythroid, megakaryocyte, ormonocyte lineage cells. Myeloproliferative disorders (MPD) includepolycythemia vera (PV), essential thrombocythemia (ET), myeloidmetaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML),chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome(HES), juvenile myelomonocytic leukemia (JMML) and systemic mast celldisease (SMCD). It has been suggested that abnormalities in signaltransduction mechanisms, including constitutive activation of proteintyrosine kinases, initiate MPD.

JAK3 associates with the common gamma chain of the extracellularreceptors for the following interleukins: IL-2, IL-4, IL-7, IL-9 andIL-15. A JAK3 deficiency is associated with an immune compromised (SCID)phenotype in both rodents and humans. The SCID phenotype of JAK3−/−mammals and the lymphoid cell specific expression of JAK3 are twofavorable attributes of a target for an immune suppressant. Datasuggests that inhibitors of JAK3 could impede T-cell activation andprevent rejection of grafts following transplant surgery, or to providetherapeutic benefit to patients suffering autoimmune disorders.

SUMMARY OF THE INVENTION

The instant invention provides for compounds that inhibit mammalian JAKkinases (such as JAK1, JAK2, JAK3 and TYK2). The invention also providesfor compositions comprising such inhibitory compounds and methods ofinhibiting the activity of JAK1, JAK2, JAK3 and TYK2 by administeringthe compound to a patient in need of treatment for myeloproliferativedisorders or cancer. One embodiment of the invention is illustrated by acompound of formula I, and the pharmaceutically acceptable salts andstereoisomers thereof:

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides for compounds that inhibit the four knownmammalian JAK kinases (JAK1, JAK2, JAK3 and TYK2). The invention alsoprovides for compositions comprising such inhibitory compounds andmethods of inhibiting the activity of JAK1, JAK2, JAK3 and TYK2 byadministering the compound to a patient in need of treatment formyeloproliferative disorders or cancer. One embodiment of the inventionis illustrated by a compound of formula I:

wherein R¹ is aryl, which is optionally substituted with one to threesubstituents independently selected from the group consisting of halo,hydroxyl, cyano, SO₂R³, C₁₋₆ alkyl and heterocyclyl; wherein said alkylgroup is optionally substituted with one to three halo, hydroxyl orcyano, and said heterocyclyl group is optionally substituted on eitherthe carbon or heteroatom with one to three halo, hydroxyl or oxo;R² is aryl or heteroaryl, wherein said aryl and heteroaryl groups areoptionally substituted on either the carbon or heteroatom with one tothree substituents selected from the group consisting of halo, cyano,C₁₋₆ alkyl, heterocyclyl, (C═O)heterocyclyl, (C═O)R³, (C═O)OR³,(C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴, NR³SO₂R⁴ and SO₂(heterocyclyl); whereinsaid alkyl group is optionally substituted with one to three halo,hydroxyl, O(C═O)NR³R⁴, heteroaryl, heterocyclyl or NR³R⁴; saidheterocyclyl groups are optionally substituted on either the carbon orheteroatom with one to two groups independently selected from the groupconsisting of C₁₋₃ alkyl, hydroxyl and oxo; and said heteroaryl groupsare optionally substituted on either the carbon or heteroatom with C₁₋₃alkyl;R³ is hydrogen or C₁₋₆ alkyl;R⁴ is hydrogen or C₁₋₆ alkyl;or a pharmaceutically acceptable salt thereof.

In an embodiment of the invention, R¹ is aryl, wherein said aryl groupis substituted with one to three substituents independently selectedfrom the group consisting of halo and C₁₋₆ alkyl, wherein said alkylgroup is optionally substituted with hydroxyl. In a class of theinvention, R¹ is phenyl, wherein said phenyl group is substituted withone to three halo.

In an embodiment of the invention, R² is aryl or heteroaryl, whereinsaid aryl and heteroaryl groups are optionally substituted on either thecarbon or heteroatom with one to three substituents selected from thegroup consisting of C₁₋₆ alkyl, (C═O)heterocyclyl, (C═O)NR³R⁴, SO₂R³,SO₂NR³R⁴ and SO₂(heterocyclyl); wherein said alkyl group is optionallysubstituted with hydroxyl, heterocyclyl or NR³R⁴; said heterocyclylgroups are optionally substituted on either the carbon or heteroatomwith one to two groups independently selected from the group consistingof C₁₋₃ alkyl and oxo. In a class of the invention, R² is aryl, whereinsaid aryl group is optionally substituted with one to three substituentsselected from the group consisting of C₁₋₆ alkyl, (C═O)heterocyclyl,(C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴ and SO₂(heterocyclyl); wherein said alkylgroup is optionally substituted with hydroxyl, heterocyclyl or NR³R⁴;said heterocyclyl groups are optionally substituted on either the carbonor heteroatom with one to two groups independently selected from thegroup consisting of C₁₋₃ alkyl and oxo. In a subclass of the invention,R² is phenyl, wherein said phenyl group is optionally substituted withone to three substituents selected from the group consisting of C₁₋₆alkyl, (C═O)heterocyclyl, (C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴ andSO₂(heterocyclyl); wherein said alkyl group is optionally substitutedwith hydroxyl or heterocyclyl; said heterocyclyl groups are optionallysubstituted on either the carbon or heteroatom with one to two groupsindependently selected from the group consisting of C₁₋₃ alkyl, and oxo.

Reference to the preferred embodiments set forth above is meant toinclude all combinations of particular and preferred groups unlessstated otherwise.

Specific embodiments of the present invention include, but are notlimited to:

-   3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(methylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-{[2-(hydroxymethyl)pyridin-4-yl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   3-{[4-(morpholin-4-ylcarbonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   3-{[4-(morpholin-4-ylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(dimethylamino)sulfonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(dimethylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-anilino-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(1,1-dioxidothiomorpholin-4-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-({4-[(dimethylamino)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;-   3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;-   1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;-   1-[4-(methylsulfonyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;-   1-(4-tert-butylphenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;-   1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;    or a pharmaceutically acceptable salt thereof.

Also included within the scope of the present invention is apharmaceutical composition which is comprised of a compound of Formula Ias described above and a pharmaceutically acceptable carrier. Theinvention is also contemplated to encompass a pharmaceutical compositionwhich is comprised of a pharmaceutically acceptable carrier and any ofthe compounds specifically disclosed in the present application. Theseand other aspects of the invention will be apparent from the teachingscontained herein.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, all such stereoisomers beingincluded in the present invention.

In addition, the compounds disclosed herein may exist as tautomers andboth tautomeric forms are intended to be encompassed by the scope of theinvention, even though only one tautomeric structure is depicted. Forexample the following is within the scope of the instant invention:

Many heteroaryl groups, such as imidazoles, exist as a mixture of 1H/2Htautomers. The tautomeric forms of these heteroaryl moieties are alsowithin the scope of the instant invention.

When any variable (e.g. R³, etc.) occurs more than one time in anyconstituent, its definition on each occurrence is independent at everyother occurrence. Also, combinations of substituents and variables arepermissible only if such combinations result in stable compounds. Linesdrawn into the ring systems from substituents represent that theindicated bond may be attached to any of the substitutable ring atoms.If the ring system is bicyclic, it is intended that the bond be attachedto any of the suitable atoms on either ring of the bicyclic moiety.

It is understood that one or more silicon (Si) atoms can be incorporatedinto the compounds of the instant invention in place of one or morecarbon atoms by one of ordinary skill in the art to provide compoundsthat are chemically stable and that can be readily synthesized bytechniques known in the art from readily available starting materials.Carbon and silicon differ in their covalent radius leading todifferences in bond distance and the steric arrangement when comparinganalogous C-element and Si-element bonds. These differences lead tosubtle changes in the size and shape of silicon-containing compoundswhen compared to carbon. One of ordinary skill in the art wouldunderstand that size and shape differences can lead to subtle ordramatic changes in potency, solubility, lack of off target activity,packaging properties, and so on. (Diass, J. O. et al. Organometallics(2006) 5:1188-1198; Showell, G. A. et al. Bioorganic & MedicinalChemistry Letters (2006) 16:2555-2558).

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrase “optionally substituted with one or more substituents” shouldbe taken to be equivalent to the phrase “optionally substituted with atleast one substituent” and in such cases the preferred embodiment willhave from zero to four substituents, and the more preferred embodimentwill have from zero to three substituents.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁₋₆, as in “(C₁₋₆)alkyl”is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in alinear or branched arrange-ment. For example, “(C₁₋₆)alkyl” specificallyincludes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl,pentyl, hexyl, and so on.

The term “haloalkyl” means an alkyl radical as defined above, unlessotherwise specified, that is substituted with one to five, preferablyone to three halogen. Representative examples include, but are notlimited to trifluoromethyl, dichloroethyl, and the like.

The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. For example,“cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on.

“Alkoxy” represents either a cyclic or non-cyclic alkyl group ofindicated number of carbon atoms attached through an oxygen bridge.“Alkoxy” therefore encompasses the definitions of alkyl and cycloalkylabove.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydro-naphthyl, indanyl and biphenyl. In cases where thearyl substituent is bicyclic and one ring is non-aromatic, it isunderstood that attachment is via the aromatic ring.

The term “heteroaryl,” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition ofheterocycle below, “heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Suchheteraoaryl moieties include but are not limited to: 2-benzimidazolyl,2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 1-isoquinolinyl,3-isoquinolinyl, 4-isoquinolinyl, dihydroimidazopyrazinyl anddihydrooxozolopyridinyl.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. Further examples of “heterocyclyl”include, but are not limited to the following: azabicyclohexyl,azaphosphinyl, azaspiroheptyl, benzoimidazolyl, benzoimidazolonyl,benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl,benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl,dioxidothiomorpholinyl, furanyl, imidazolyl, indolinyl, indolyl,indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl,oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl,tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl,azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, oxadiazaspirodecyl, tetrahydrofuranyl, andtetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclylsubstituent can occur via a carbon atom or via a heteroatom.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro (Cl), fluoro (F), bromo (Br) andiodo (I).

Included in the instant invention is the free form of compounds of theinstant invention, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific compoundsexemplified herein are the protonated salts of amine compounds. The term“free form” refers to the amine compounds in non-salt form. Theencompassed pharmaceutically acceptable salts not only include theisolated salts exemplified for the specific compounds described herein,but also all the typical pharmaceutically acceptable salts of the freeform of compounds of the instant invention. The free form of thespecific salt compounds described may be isolated using techniques knownin the art. For example, the free form may be regenerated by treatingthe salt with a suitable dilute aqueous base solution such as diluteaqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. Thefree forms may differ from their respective salt forms somewhat incertain physical properties, such as solubility in polar solvents, butthe acid and base salts are otherwise pharmaceutically equivalent totheir respective free forms for purposes of the invention.

The pharmaceutically acceptable salts of the instant compounds can besynthesized from the compounds of this invention which contain a basicor acidic moiety by conventional chemical methods. Generally, the saltsof the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the compounds of thisinvention include the conventional non-toxic salts of the compounds ofthis invention as formed by reacting a basic instant compound with aninorganic or organic acid. For example, conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, aswell as salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic(TFA) and the like.

When the compound of the present invention is acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glutamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

Utility

The compounds of the present invention are inhibitors of JAK1, JAK2, JAK3, and TYK2, and are therefore useful to treat or preventmyeloproliferative disorders or cancer in mammals, preferably humans.

An embodiment of the invention provides a method for inhibiting JAK1tyrosine kinase, comprising administering to the mammal atherapeutically effective amount of any of the compounds or any of thepharmaceutical compositions described above.

An embodiment of the invention provides a method for inhibiting JAK2tyrosine kinase, comprising administering to the mammal atherapeutically effective amount of any of the compounds or any of thepharmaceutical compositions described above.

An embodiment of the invention provides a method for inhibiting wildtype or mutant JAK2 tyrosine kinase, comprising administering to themammal a therapeutically effective amount of any of the compounds or anyof the pharmaceutical compositions described above.

An embodiment of the invention provides a method for inhibitingJAK2V617F tyrosine kinase, comprising administering to the mammal atherapeutically effective amount of any of the compounds or any of thepharmaceutical compositions described above.

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of myeloproliferative disorder(s).Myeloproliferative disorders that may be treated include polycythemiavera (PV), essential thrombocythemia (ET), myeloid metaplasia withmyelofibrosis (MMM), chronic myelogenous leukemia (CML), myelomonocyticleukemia (CMML), hypereosinophilic syndrome (HES), juvenilemyelomonocytic leukemia (JMML), and systemic mast cell disease (SMCD).

It is known in the literature that inhibitors of JAK2 are useful in thetreatment and/or prevention of myeloproliferative disorders. See, e.g.,Tefferi, A. and Gilliland, D. G. Mayo Clin. Proc. 80(7): 947-958 (2005);Fernandez-Luna, J. L. et al. Haematologica 83(2): 97-98 (1998);Harrison, C. N. Br. J. Haematol. 130(2): 153-165 (2005); Leukemia (2005)19, 1843-1844; and Tefferi, A. and Barbui, T. Mayo Clin. Proc. 80(9):1220-1232 (2005).

The compounds, compositions and methods provided herein are also deemeduseful for the treatment of cancer. Cancers that may be treated by thecompounds, compositions and methods of the invention include, but arenot limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma), colon, colorectal, rectal; Genitourinary tract:kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma),testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma; and Adrenal glands: neuroblastoma. Thus, the term“cancerous cell” as provided herein, includes a cell afflicted by anyone of the above-identified conditions.

The compounds, compositions and methods of the invention may also beuseful in treating the following disease states: keloids and psoriasis.

Cancers that may be treated by the compounds, compositions and methodsof the invention include, but are not limited to: breast, prostate,colon, colorectal, lung, brain, testicular, stomach, pancrease, skin,small intestine, large intestine, throat, head and neck, oral, bone,liver, bladder, kidney, thyroid and blood.

Cancers that may be treated by the compounds, compositions and methodsof the invention include: breast, prostate, colon, ovarian, colorectaland lung (non-small cell lung).

Cancers that may be treated by the compounds, compositions and methodsof the invention include: breast, colon, colorectal and lung.

Cancers that may be treated by the compounds, compositions and methodsof the invention include: lymphoma and leukemia.

Further included within the scope of the invention is a method oftreating or preventing a disease in which angiogenesis is implicated,which is comprised of administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of thepresent invention. Ocular neovascular diseases are an example ofconditions where much of the resulting tissue damage can be attributedto aberrant infiltration of blood vessels in the eye (see WO 00/30651,published 2 Jun. 2000). The undesirable infiltration can be triggered byischemic retinopathy, such as that resulting from diabetic retinopathy,retinopathy of prematurity, retinal vein occlusions, etc., or bydegenerative diseases, such as the choroidal neovascularization observedin age-related macular degeneration. Inhibiting the growth of bloodvessels by administration of the present compounds would thereforeprevent the infiltration of blood vessels and prevent or treat diseaseswhere angiogenesis is implicated, such as ocular diseases like retinalvascularization, diabetic retinopathy, age-related macular degeneration,and the like.

Further included within the scope of the invention is a method oftreating or preventing a non-malignant disease in which angiogenesis isimplicated, including but not limited to: ocular diseases (such as,retinal vascularization, diabetic retinopathy and age-related maculardegeneration), atherosclerosis, arthritis, psoriasis, obesity andAlzheimer's disease (Dredge et al., Expert Opin. Biol. Ther. (2002)2(8):953-966). In another embodiment, a method of treating or preventinga disease in which angiogenesis is implicated includes: ocular diseases(such as, retinal vascularization, diabetic retinopathy and age-relatedmacular degeneration), atherosclerosis, arthritis and psoriasis.

Further included within the scope of the invention is a method oftreating hyperproliferative disorders such as restenosis, inflammation,autoimmune diseases and allergy/asthma.

Further included within the scope of the instant invention is the use ofthe instant compounds to coat stents and therefore the use of theinstant compounds on coated stents for the treatment and/or preventionof restenosis (WO03/032809).

Further included within the scope of the instant invention is the use ofthe instant compounds for the treatment and/or prevention ofosteoarthritis (WO03/035048).

Further included within the scope of the invention is a method oftreating hypoinsulinism.

An embodiment of the invention provides a method for inhibiting JAK3tyrosine kinase, comprising administering to the mammal atherapeutically effective amount of any of the compounds or any of thepharmaceutical compositions described above.

An embodiment of the invention provides a method for inhibiting TYK2tyrosine kinase, comprising administering to the mammal atherapeutically effective amount of any of the compounds or any of thepharmaceutical compositions described above.

Exemplifying the invention is the use of any of the compounds describedabove in the preparation of a medicament for the treatment and/orprevention of osteoporosis in a mammal in need thereof. Still furtherexemplifying the invention is the use of any of the compounds describedabove in the preparation of a medicament for the treatment and/orprevention of: bone loss, bone resorption, bone fractures, metastaticbone disease and/or disorders related to cathepsin functioning.

The compounds of this invention may be administered to mammals,including humans, either alone or, in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintravenous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropylmethyl-cellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsion. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of sterile injectableaqueous solutions. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of the instant invention may also be administered in the formof suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter, glycerinatedgelatin, hydrogenated vegetable oils, mixtures of polyethylene glycolsof various molecular weights and fatty acid esters of polyethyleneglycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of the instant invention are employed.(For purposes of this application, topical application shall includemouth washes and gargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Compounds of the presentinvention may also be delivered as a suppository employing bases such ascocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds of the present invention may alsobe coupled with soluble polymers as targetable drug carriers. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polyactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcrosslinked or amphipathic block copolymers of hydro gels.

When a composition according to this invention is administered into ahuman subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

In an embodiment, a suitable amount of an inhibitor of JAK2 isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount of inhibitor of between about 0.1 mg/kg of bodyweight to about 60 mg/kg of body weight per day, or between 0.5 mg/kg ofbody weight to about 40 mg/kg of body weight per day. Anothertherapeutic dosage that comprises the instant composition includes fromabout 0.01 mg to about 1000 mg of inhibitor of JAK2. In anotherembodiment, the dosage comprises from about 1 mg to about 1000 mg ofinhibitor of JAK2.

The instant compounds are also useful in combination with therapeutic,chemotherapeutic and anti-cancer agents. Combinations of the presentlydisclosed compounds with therapeutic, chemotherapeutic and anti-canceragents are within the scope of the invention. Examples of such agentscan be found in Cancer Principles and Practice of Oncology by V. T.Devita and S. Hellman (editors), 6^(th) edition (Feb. 15, 2001),Lippincott Williams & Wilkins Publishers. A person of ordinary skill inthe art would be able to discern which combinations of agents would beuseful based on the particular characteristics of the drugs and thecancer involved. Such agents include the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic/cytostatic agents, antiproliferative agents, prenyl-proteintransferase inhibitors, HMG-CoA reductase inhibitors and otherangiogenesis inhibitors, HIV protease inhibitors, reverse transcriptaseinhibitors, inhibitors of cell proliferation and survival signaling,bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretaseinhibitors, agents that interfere with receptor tyrosine kinases (RTKs)and agents that interfere with cell cycle checkpoints. The instantcompounds are particularly useful when co-administered with radiationtherapy.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell myosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, histone deacetylase inhibitors,inhibitors of kinases involved in mitotic progression, inhibitors ofkinases involved in growth factor and cytokine signal transductionpathways, antimetabolites, biological response modifiers,hormonal/anti-hormonal therapeutic agents, haematopoietic growthfactors, monoclonal antibody targeted therapeutic agents, topoisomeraseinhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, andaurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to,sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin,altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine,nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine,improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755,4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and mTORinhibitors (such as Wyeth's CCI-779).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited tolactacystin, MLN-341 (Velcade) and carfilzomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797. In an embodiment the epothilones are notincluded in the microtubule inhibitors/microtubule-stabilising agents.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]aeridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in Publications WO03/039460,WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678,WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417,WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638,WO05/019206, WO05/019205, WO05/018547, WO05/017190, US2005/0176776. Inan embodiment inhibitors of mitotic kinesins include, but are notlimited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E,inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are notlimited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Furtherreference to other histone deacetylase inhibitors may be found in thefollowing manuscript; Miller, T. A. et al. J. Med. Chem.46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” isVX-680.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine,3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) andcerivastatin (also known as rivastatin and BAYCHOL®; see U.S. Pat. No.5,177,080). The structural formulas of these and additional HMG-CoAreductase inhibitors that may be used in the instant methods aredescribed at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”,Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefor the use of such salts, esters,open-acid and lactone forms is included within the scope of thisinvention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer tocompounds that inhibit RTKs and therefore mechanisms involved inoncogenesis and tumor progression. Such agents include inhibitors ofc-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors ofRTKs as described by Burne-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” referto compounds that inhibit signal transduction cascades downstream ofcell surface receptors. Such agents include inhibitors ofserine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in WO 02/083064, WO 02/083139, WO 02/083140, US2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279,WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US2005/43361, 60/734,188, 60/652,737, 60/670,469), inhibitors of Rafkinase (for example BAY-43-9006), inhibitors of MEK (for example CI-1040and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), andinhibitors of PI3K (for example LY294002).

As described above, the combinations with NSAID's are directed to theuse of NSAID's which are potent COX-2 inhibiting agents. For purposes ofthis specification an NSAID is potent if it possesses an IC₅₀ for theinhibition of COX-2 of 1 μM or less as measured by cell or microsomalassays.

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, U.S. Pat. No.5,861,419, U.S. Pat. No. 6,001,843, U.S. Pat. No. 6,020,343, U.S. Pat.No. 5,409,944, U.S. Pat. No. 5,436,265, U.S. Pat. No. 5,536,752, U.S.Pat. No. 5,550,142, U.S. Pat. No. 5,604,260, U.S. Pat. No. 5,698,584,U.S. Pat. No. 5,710,140, WO 94/15932, U.S. Pat. No. 5,344,991, U.S. Pat.No. 5,134,142, U.S. Pat. No. 5,380,738, U.S. Pat. No. 5,393,790, U.S.Pat. No. 5,466,823,U.S. Pat. No. 5,633,272 and U.S. Pat. No. 5,932,598,all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone;and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; ora pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to, the following: parecoxib, BEXTRA® and CELEBREX® or apharmaceutically acceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the αvβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, ST1571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-6 are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem., 1999; 274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), andinterferon gamma (J. Immunol. 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In another embodiment, conjunctive therapywith an anti-emesis agent selected from a neurokinin-1 receptorantagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosedfor the treatment or prevention of emesis that may result uponadministration of the instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93106099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing cancer, including bone cancer, in combination withbisphosphonates (understood to include bisphosphonates, diphosphonates,bisphosphonic acids and diphosphonic acids). Examples of bisphosphonatesinclude but are not limited to: etidronate (Didronel), pamidronate(Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate(Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate,EB-1053, minodronate, neridronate, piridronate and tiludronate includingany and all pharmaceutically acceptable salts, derivatives, hydrates andmixtures thereof.

A compound of the instant invention may also be useful for treating orpreventing breast cancer in combination with aromatase inhibitors.Examples of aromatase inhibitors include but are not limited to:anastrozole, letrozole and exemestane.

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with siRNA therapeutics.

The compounds of the instant invention may also be administered incombination with γ-secretase inhibitors and/or inhibitors of NOTCHsignaling. Such inhibitors include compounds described in WO 01/90084,WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370,WO 2005/030731, WO 2005/014553, U.S. Ser. No. 10/957,251, WO2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137,WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO02/47671 (including LY-450139).

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with inhibitors of Akt. Such inhibitorsinclude compounds described in, but not limited to, the followingpublications: WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432,WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394,WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734,188,60/652,737, 60/670,469.

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with PARP inhibitors.

A compound of the instant invention may also be useful for treatingcancer in combination with the following therapeutic agents: abarelix(Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®);Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol(Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole(Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®);azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotene capsules(Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®);bortezomib (Velcade®); busulfan intravenous (Busulfex®); busulfan oral(Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®);carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine(Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®);celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®);cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine(Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide(Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileuldndiftitox (Ontak®); dexrazoxane (Zinecard®); docetaxel (Taxotere®);doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®);doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®);dromostanolone propionate (Dromostanolone®); dromostanolone propionate(Masterone Injection®); Elliott's B Solution (Elliott's B Solution®);epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®);estramustine (Emcyte®); etoposide phosphate (Etopophos®); etoposide,VP-16 (Vepesid®); exemestane (Aromasin®); Filgrastim (Neupogen®);floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®);fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib(Iressa®); gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®);goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®);histrelin acetate (Histrelin implant®); hydroxyurea (Hydrea®);Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide(IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®);Interferon alfa-2b (Intron A®); irinotecan (Camptosar®); lenalidomide(Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®,Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®);lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®);megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®);mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnextabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C(Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®);nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®);Nofetumomab (Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®);paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-boundparticles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®);pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®);Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin(Nipent®); pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®);porfimer sodium (Photofrin®); procarbazine (Matulane®); quinacrine(Atabrine®); Rasburicase (Elitek®); Rituximab (Rituxan®); sargramostim(Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin(Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen(Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®);testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa(Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab(Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®); Trastuzumab(Herceptin®); tretinoin, ATRA (Vesanoid®); Uracil Mustard (UracilMustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®);vincristine (Oncovin®); vinorelbine (Navelbine®); and zoledronate(Zometa®).

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: an estrogen receptor modulator, an androgen receptormodulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent,an antiproliferative agent, a prenyl-protein transferase inhibitor, anHMG-CoA reductase inhibitor, an HIV protease inhibitor, a reversetranscriptase inhibitor, an angiogenesis inhibitor, PPAR-γ agonists,PPAR-δ agonists, an inhibitor of inherent multidrug resistance, ananti-emetic agent, an agent useful in the treatment of anemia, an agentuseful in the treatment of neutropenia, an immunologic-enhancing drug,an inhibitor of cell proliferation and survival signaling, abisphosphonate, an aromatase inhibitor, an siRNA therapeutic,γ-secretase inhibitors, agents that interfere with receptor tyrosinekinases (RTKs), an agent that interferes with a cell cycle checkpointand any of the therapeutic agents listed above.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of the instant invention in combination with radiation therapyand/or in combination with a second compound selected from: an estrogenreceptor modulator, an androgen receptor modulator, a retinoid receptormodulator, a cytotoxiccytostatic agent, an antiproliferative agent, aprenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, anHIV protease inhibitor, a reverse transcriptase inhibitor, anangiogenesis inhibitor, PPAR-γ agonists, PPAR-δ agonists, an inhibitorof inherent multidrug resistance, an anti-emetic agent, an agent usefulin the treatment of anemia, an agent useful in the treatment ofneutropenia, an immunologic-enhancing drug, an inhibitor of cellproliferation and survival signaling, a bisphosphonate, an aromataseinhibitor, an siRNA therapeutic, γ-secretase inhibitors, agents thatinterfere with receptor tyrosine kinases (RTKs), an agent thatinterferes with a cell cycle checkpoint and any of the therapeuticagents listed above.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of the instant invention and a secondcompound selected from: an estrogen receptor modulator, an androgenreceptor modulator, a retinoid receptor modulator, acytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-proteintransferase inhibitor, an HMG-CoA reductase inhibitor, an HIV proteaseinhibitor; a reverse transcriptase inhibitor, an angiogenesis inhibitor,a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of cell proliferationand survival signaling, a bisphosphonate, an aromatase inhibitor, ansiRNA therapeutic, γ-secretase inhibitors, agents that interfere withreceptor tyrosine kinases (RTKs), an agent that interferes with a cellcycle checkpoint and any of the therapeutic agents listed above.

All patents, publications and pending patent applications identified arehereby incorporated by reference.

The abbreviations used herein have the following tabulated meanings.Abbreviations not tabulated below have their meanings as commonly usedunless specifically stated otherwise.

Ac acetyl Bn benzyl CHCl₃ chloroform Cu(OAc)₂ copper acetate DCMdichloromethane DIPEA diisopropylethylamine DMF N,N-dimethylformamideDMSO dimethyl sulfoxide EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc ethyl acetate EtOH ethanol HCl hydrogenchloride H₂O₂ hydrogen peroxide H₂SO₄ hydrogen sulfide K₂CO₃ potassiumcarbonate MeOH methanol MgSO₄ magnesium sulfide Ms methanesulfonyl =mesyl = SO₂Me NH₄Cl ammonium chloride NaOH hydrazine NaHCO₃N-methylmorpholine-N-oxide NSAID non-steroidal anti-inflammatory drugPd₂dba₃ tris(dibenzylideneacetone)dipalladium(0) Pd(PPh₃)₄tetrakis(triphenylphosphine)palladium (I) Ph phenyl PhB(OH)₂ phenylboronic acid POCl₃ phorphorous oxychloride r.t. room temperature Rac.racemic SEM 2-(trimethylsilyl)ethoxymethoxy TEA triethylamine TFAtrifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography

Alkyl Group Abbreviations

Me Methyl Et Ethyl n-Pr normal propyl i-Pr isopropyl n-Bu normal butyli-Bu isobutyl s-Bu secondary butyl t-Bu tertiary butyl c-Pr cyclopropylc-Bu cyclobutyl c-Pen cyclopentyl c-Hex cyclohexyl

The compounds of the present invention can be prepared according to thefollowing general schemes, using appropriate materials, and are furtherexemplified by the subsequent specific examples. The compoundsillustrated in the examples are not, however, to be construed as formingthe only genus that is considered as the invention. The illustrativeExamples below, therefore, are not limited by the compounds listed or byany particular substituents employed for illustrative purposes. Thoseskilled in the art will readily understand that known variations of theconditions and processes of the following preparative procedures can beused to prepare these compounds. All temperatures are degrees Celsiusunless otherwise noted.

Methods of Synthesis Method 1

General procedures to prepare compounds of the instant invention aredescribed in Scheme 1. Hydrazide I can be condensed with(ethoxymethylene)malononitrile to yield pyrazole enamine II. The enaminecan be hydrolyzed under acidic conditions to provide 3-amino pyrazolesIII and IV. Cyano pyrazole III can be subsequently hydrated to provideamido pyrazole IV. The 3-amino pyrazole IV can be elaborated to thefinal product pyrazole V through a palladium catalyzed coupling with anappropriate optionally substituted halogenated (hetero)aromatic.

Method 2

General procedures to prepare compounds of the instant invention aredescribed in Scheme 2. 3-amino-1H-pyrazole-4-carbonitrile VI can bearylated with phenylboronic acid to yield a mixture of anilino pyrazoleVII-A and 3-amino pyrazole III. The 3-amino pyrazole III can beelaborated to pyrazole VII through a palladium catalyzed coupling withan appropriate optionally substituted halogenated (hetero)aromatic. Thefinal product pyrazole V can be obtained by hydration of cyano pyrazoleVII.

Method 3

General procedures to prepare compounds of the instant invention aredescribed in Scheme 3. 3-amino-1H-pyrazole-4-carbonitrile VI can bereacted with SEM-C1 to yield a mixture 3-amino pyrazoles VIIIA andVIIIB. Regioisomer VIIIB can be arylated to give pyrazole IX through apalladium catalyzed coupling with an appropriate optionally substitutedhalogenated (hetero)aromatic. The SEM group can be removed by acidhydrolysis to yield pyrazole X. The 3-arylamino pyrazole X can beelaborated to pyrazole VII through a copper catalyzed arylation with anappropriate optionally substituted arylboronic acid. The final productpyrazole V can be obtained by hydration of cyano pyrazole VII.

The invention will now be illustrated in the following non-limitingexamples in which, unless otherwise stated:1. All final products were analyzed by NMR, LCMS.2. Intermediates were analyzed by NMR and/or TLC and/or LCMS.3. Most compounds were purified by flash chromatography on silica gel,reverse phase HPLC, recrystallization, and/or swish (suspension in asolvent followed by filtration of the solid).4. The course of the reactions was followed by thin layer chromatography(TLC) and/or LCMS and reaction times are given for illustration only.

Example 1

3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

Step 1.{[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]methylene}malononitrile

N′-phenylacetohydrazide (5 g, 33.3 mmol) and(ethoxymethylene)malononitrile (8.13 g, 66.6 mmol) were stirred inphosphorus oxychloride (6.21 ml, 66.6 mmol) at 100° C. for 30 minutes.Room temperature was attained, water was added and the dark solidcollected by filtration. The residue was triturated in EtOH to give{[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]methylene}malononitrile as abeige solid (batch 1). The filtrate was concentrated in vacuo and theresidue purified by MPLC (6-50% EtOAc-hexanes) to give{[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]methylene}malononitrile as anorange solid after recrystallizing from EtOAc-hexanes (batch 2).

LRMS (APCI) calc'd for (C₁₄H₉N₆) [M+H]⁺: 261, Found: 261.

Step 2. 3-amino-1-phenyl-1H-pyrazole-4-carbonitrile and3-amino-1-phenyl-1H-pyrazole-4-carboxamide

{[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]methylene}malononitrile (1.07g, 4.11 mmol) was stirred in refluxing dioxane (80 mL)/2 N HCl (80 mL)for 3 days. Room temperature was attained and the products extractedinto EtOAc (×2). The combined organic extracts were washed with brine,dried over MgSO₄ and concentrated in vacuo. Purification of the residueby MPLC (12-100% EtOAc-hexanes followed by 0-10% MeOH-EtOAc) gave3-amino-1-phenyl-1H-pyrazole-4-carbonitrile as a beige solid (A) and3-amino-1-phenyl-1H-pyrazole-4-carboxamide as a brown solid (B).

A—LRMS (APCI) calc'd for (C₁₀H₉N₄) [M+H]⁺: 185, Found: 185.

B—LRMS (APCI) calc'd for (C₁₀H₁₁N₄O) [M+H]⁺: 203, Found: 203.

Step 3. 3-3-amino-1-phenyl-1H-pyrazole-4-carboxamide

3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (0.34 g, 1.846 mmol) wasstirred in 95% H₂SO₄ (10 mL) at 50° C. for 3 hours. Room temperature wasattained, water was added and the products extracted into EtOAc (×3).The aqueous phase was neutralised with 2 N NaOH and the productsextracted into DCM (×2) followed by 20% MeOH-DCM (×4). The combinedorganic extracts were dried over MgSO₄ and concentrated in vacuo. Theresidue was triturated in EtOAc to give3-amino-1-phenyl-1H-pyrazole-4-carboxamide as a pale yellow solid.

LRMS (APCI) calc'd for (C₁₀H₁₁N₄O) [M+H]⁺: 203, Found: 203.

Step 4.3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

3-amino-1-phenyl-1H-pyrazole-4-carboxamide (50 mg, 0.247 mmol),4-bromophenyl methyl sulfone (58.1 mg, 0.247 mmol), Pd₂ dba₃ (22.64 mg,0.025 mmol), K₂CO₃ (37.6 mg, 0.272 mmol) and X-Phos (58.9 mg, 0.124mmol) were added to a 5 mL microwave vial. Degassed EtOH (0.75 mL) wasadded and the vial evacuated and back-filled with N₂ (×3). The resultingmixture was stirred at 100° C. for 4 hours. Room temperature wasattained, MeOH was added and the solvent removed in vacuo while loadingonto silica. Purification of the residue by MPLC (0-6% MeOH—CHCl₃) gave3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamideas a pale yellow solid after triturating in DCM.

LRMS (APCI) calc'd for (C₁₇H₁₇N₄O₃S) [M+H]⁺: 357, Found: 357. JAK2IC₅₀=170 nM.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples.

TABLE 1 JAK2 Example Characterization IC₅₀ # Structure Compound Name[M + H]⁺ (nM) 2

3-({4- [(methylamino) carbonyl]phenyl} amino)-1-phenyl- 1H-pyrazole-4-carboxamide Calc'd 336, found 336 160 3

3-{[2- (hydroxymethyl) pyridin-4-yl] amino}-1-phenyl- 1H-pyrazole-4-carboxamide Calc'd 310, found 310 860

Example 4

3-{[4-(morpholin-4-ylcarbonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 4(4-bromobenzoyl)morpholine

4-bromobenzoyl chloride (5 g, 22.78 mmol) was taken up in THF (75 mL)and cooled to 0° C. A solution of morpholine (2.084 mL, 23.92 mmol) andDIPEA (4.38 mL, 25.06 mmol) in THF (25 mL) was added dropwise and theresulting mixture stirred at 0° C. for 3 hours. Water as added and theproducts extracted into EtOAc (×2). The combined organic extracts werewashed with brine, dried over MgSO₄ and concentrated in vacuo to give4-(4-bromobenzoyl)morpholine as a pale yellow gum.

LRMS (APCI) calc'd for (C₁₁H₁₃BrNO₂) [M+H]⁺: 270, 272, Found: 270, 272.

Step 2.3-{[4-(morpholin-4-ylcarbonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

3-{[4-(morpholin-4-ylcarbonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 1 Step 4using 3-amino-1-phenyl-1H-pyrazole-4-carboxamide (52 mg, 0.257 mmol) and4-(4-bromobenzoyl)morpholine (69.5 mg, 0.257 mmol) as the startingmaterials.

LRMS (APCI) calc'd for (C₂₁H₂₂N₅O₃) [M+H]⁺: 392, Found: 392. JAK2IC₅₀=94 nM.

Example 5

3-{[4-(morpholin-4-ylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 4-[(4-bromophenyl)sulfonyl]morpholine

Morpholine (58.2 mg, 0.668 mmol) and triethylamine (116 μl, 0.835 mmol)were placed in a round-bottom flask with a stir bar. Dry dichloromethane(5870 μL) was then added and the solution cooled to 0° C.4-bromobenzenesulfonyl chloride (150 mg, 0.556 mmol) was added and thereaction was allowed to warm to room temperature overnight. It wasquenched with water and extracted in ethyl acetate. The organic layerswere washed with aqueous sodium bicarbonate, dried over magnesiumsulfate, filtered, and concentrated under reduced pressure to give4-[(4-bromophenyl)sulfonyl]morpholine as a white solid.

LRMS (APCI) calc'd for (C₁₁H₁₃BrNO₂) [M+H]⁺: 270, 272, Found: 270, 272.

Step 2.3-{[4-(morpholin-4-ylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

3-{[4-(morpholin-4-ylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 1 Step 4using 3-amino-1-phenyl-1H-pyrazole-4-carboxamide (34 mg, 0.168 mmol) and4-[(4-bromophenyl)sulfonyl]morpholine (51.5 mg, 0.168 mmol) as thestarting materials.

LRMS (APCI) calc'd for (C₂₀H₂₂N₅O₄S) [M+H]⁺: 428, Found: 428. JAK2IC₅₀=63 nM.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples.

TABLE 2 JAK2 Characterization IC₅₀ Example # Structure Compound Name[M + H]⁺ (nM) 6

3-({4- [(dimethylamino) sulfonyl]phenyl} amino)-1-phenyl- 1H-pyrazole-4-carboxamide Calc'd 386, found 386 42

Example 7

3-({4-[(dimethylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 3-anilino-1-phenyl-1H-pyrazole-4-carbonitrile and3-amino-1-phenyl-1H-pyrazole-4-carbonitrile

3-amino-1H-pyrazole-4-carbonitrile (200 mg, 1.850 mmol), copper(II)acetate (504 mg, 2.78 mmol), phenylboronic acid (451 mg, 3.70 mmol) andpyridine (0.599 mL, 7.40 mmol) were stirred in DCM (20 mL) at roomtemperature overnight. 2 N HCl was added and the products extracted inEtOAc (×2). The combined organic extracts were washed with brine, driedover MgSO₄ and concentrated in vacuo. Purification of the residue byMPLC (2-30% EtOAc-hexanes) gave3-anilino-1-phenyl-1H-pyrazole-4-carbonitrile (A) as a yellow solid and3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (B) as a pale yellow solid.5-amino-1-phenyl-1H-pyrazole-4-carbonitrile was also isolated.

A—LRMS (APCI) calc'd for (C₁₆H₁₃N₄) [M+H]⁺: 261, Found: 261.

B—LRMS (APCI) calc'd for (C₁₀H₉N₄) [M+H]⁺: 185, Found: 185.

Step 2.4-[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]-N,N-dimethylbenzamide

3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (60 mg, 0.326 mmol),4-bromo-N,N-dimethylbenzamide (74.3 mg, 0.326 mmol), Pd₂ dba₃ (14.91 mg,0.016 mmol), K₂CO₃ (49.5 mg, 0.358 mmol) and X-Phos (38.8 mg, 0.081mmol) were added to a 5 mL microwave vial. Tert-amyl alcohol (1 mL) wasadded and the vial was evacuated and back-filled with N₂ (×3). Theresulting mixture was stirred at 100° C. overnight. Room temperature wasattained, EtOAc was added and the solvent removed in vacuo while loadingonto silica. Purification of the residue by MPLC (0-10% MeOH—CHCl₃)followed by MPLC (12-100% EtOAc-hexanes) gave4-[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]-N,N-dimethylbenzamide as apale yellow solid.

LRMS (APCI) calc'd for (C₁₉H₁₈N₅O) [M+]⁺: 332, Found: 332.

Step 3.3-({4-[(dimethylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

4-[(4-cyano-1-phenyl-1H-pyrazol-3-yl)amino]-N,N-dimethylbenzamide (83mg, 0.250 mmol) and 5 N sodium hydroxide (0.351 mL, 1.753 mmol) weretaken up in DMSO (1 mL)/EtOH (1 mL) and warmed to 60° C. Hydrogenperoxide (0.329 mL, 3.76 mmol) was added dropwise and stirring continuedat 60° C. for 15 minutes. Room temperature was attained, water was addedand the products extracted into EtOAc (×2). The combined organicextracts were washed with brine (×2), dried over MgSO₄ and concentratedin vacuo. Purification of the residue by MPLC (25-100% EtOAc-hexanesfollowed by 0-10% MeOH-EtOAc followed by 15% MeOH—CHCl₃) gave3-({4-[(dimethylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamideas a pale yellow solid.

LRMS (APCI) calc'd for (C₁₉H₂₀N₅O₂) [M+H]⁺: 350, Found: 350. JAK2IC₅₀=56 nM.

Example 8

3-anilino-1-phenyl-1H-pyrazole-4-carboxamide

3-anilino-1-phenyl-1H-pyrazole-4-carboxamide was prepared according tothe general procedure in Example 7 Step 3 using3-anilino-1-phenyl-1H-pyrazole-4-carbonitrile (10 mg, 0.038 mmol) as thestarting material.

LRMS (APCI) calc'd for (C₁₆H₁₅N₄O) [M+H]⁺: 279, Found: 279. JAK2IC₅₀=480 nM.

Example 9

3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 4-bromobenzo 1-4-methylpiperazine

1-(4-bromobenzoyl)-4-methylpiperazine was prepared according to thegeneral procedure in Example 4 Step 1 using 4-bromobenzoyl chloride (1g, 4.56 mmol) and 1-methylpiperazine (0.532 mL, 4.78 mmol) as thestarting materials.

LRMS (APCI) calc'd for (C₁₂H₁₆BrN₂O) [M+H]⁺: 283, 285, Found: 283, 285.

Step 2.3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrile

3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrilewas prepared according to the general procedure in Example 7 Step 2using 3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (50 mg, 0.271 mmol)and 1-(4-bromobenzoyl)-4-methylpiperazine (77 mg, 0.271 mmol) as thestarting materials.

LRMS (APCI) calc'd for (C₁₂H₂₃N₆O) [M+H]⁺: 387, Found: 387.

Step 3.3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrile(90 mg, 0.233 mmol) as the starting material.

LRMS (APCI) calc'd for (C₂₂H₂₅N₆O₂) [M+H]⁺: 405, Found: 405. JAK2IC₅₀=38 nM.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples.

TABLE 3 JAK2 Characterization IC₅₀ Example # Structure Compound Name[M + H]⁺ (nM) 10

3-({4-[(1,1- dioxidothio- morpholin-4- yl)carbonyl]phenyl}amino)-1-phenyl- 1H-pyrazole-4- carboxamide Calc'd 440, found 440 13

Example 11

3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

Step 1.3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrile

3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (100 mg, 0.543 mmol),4-(4-iodobenzyl)morpholine (165 mg, 0.543 mmol), Pd₂dba₃ (24.86 mg,0.027 mmol), K₂CO₃ (83 mg, 0.597 mmol) and X-Phos (64.7 mg, 0.136 mmol)were added to a 5 mL microwave vial. Degassed tert-amyl alcohol (0.8 mL)was added and the vial was evacuated and back-filled with N₂ (×3). Theresulting mixture was stirred at 100° C. for 24 hours. Room temperaturewas attained, MeOH was added and the solvent removed in vacuo whileloading onto silica. Purification of the residue by MPLC (0-15%MeOH—CHCl₃) gave3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrileas a beige solid.

LRMS (APCI) calc'd for (C₂₁H₂₂N₅O) [M+H]⁺: 360, Found: 360.

Step 2.3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrile(81 mg, 0.225 mmol) as the starting material.

LRMS (APCI) calc'd for (C₁₇H₁₅N₄O—loss of amino fragment) [M+H]⁺: 291,Found: 291. JAK2 IC₅₀=49 nM.

Example 12

3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 4-(4-iodobenzyl)thiomorpholine 1,1-dioxide

1-(bromomethyl)-4-iodobenzene (1 g, 3.37 mmol), thiomorpholine1,1-dioxide (0.546 g, 4.04 mmol) and DIPEA (0.882 mL, 5.05 mmol) werestirred in THF (15 mL) at room temperature overnight. The solvent wasremoved in vacuo while loading onto silica. Purification of the residueby MPLC (12-100% EtOAc-hexanes) gave 4-(4-iodobenzyl)thiomorpholine1,1-dioxide as a white solid.

LRMS (APCI) calc'd for (C₁₁H₁₅INO₂S) [M+H]⁺: 352, Found: 352.

Step 2.3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrile

3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrilewas prepared according to the general procedure in Example 11 Step 1using 3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (100 mg, 0.543 mmol)and 4-(4-iodobenzyl)thiomorpholine 1,1-dioxide (191 mg, 0.543 mmol) asthe starting materials.

LRMS (APCI) calc'd for (C₁₇H₁₃N₄—loss of amino fragment) [M+H]⁺: 272,Found: 272.

Step 3.3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide

3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carbonitrile(79 mg, 0.194 mmol) as the starting material.

LRMS (APCI) calc'd for (C₂₁H₂₄N₅O₃S) [M+H]⁺: 426, Found: 426. JAK2IC₅₀=36 nM.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples.

TABLE 4 JAK2 Example Characterization IC₅₀ # Structure Compound Name[M + H]⁺ (nM) 13

3-({4- [(dimethylamino) methyl]phenyl} amino)-1-phenyl- 1H-pyrazole-4-carboxamide Calc'd 291 (loss of amino fragment), found 291 36

Example 14

3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

Step 1. 2-(4-iodophenyl)propan-2-ol

Methyl 4-iodobenzoate (10 g, 38.2 mmol) was taken up in THF (100 mL) andcooled to 0° C. Methylmagnesium bromide (38.2 ml, 114 mmol) was addeddropwise and stirring at 0° C. continued for 30 minutes. Saturated NH₄Clwas added and the products extracted into EtOAc (×2). The combinedorganic extracts were washed with brine, dried over MgSO₄ andconcentrated in vacuo. Purification of the residue by MPLC (6-50%EtOAc-hexanes) gave 2-(4-iodophenyl)propan-2-ol as a yellow oil.

LRMS (APCI) calc'd for (C₉H₁₀I—loss of water) [M+H]⁺: 245, Found: 245.

Step 2.3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrile

3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrilewas prepared according to the general procedure in Example 11 Step 1using 3-amino-1-phenyl-1H-pyrazole-4-carbonitrile (70 mg, 0.380 mmol)and 2-(4-iodophenyl)propan-2-ol (100 mg, 0.380 mmol) as the startingmaterials.

LRMS (APCI) calc'd for (C₁₉H₁₉N₄O) [M+H]⁺: 319, Found: 319.

Step 3.3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide

3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carbonitrile(36 mg, 0.113 mmol) as the starting material.

LRMS (APCI) calc'd for (C₁₉H₂₁N₄O₂) [M+H]⁺: 337, Found: 337. JAK2IC₅₀=81 nM.

Example 15

1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide

Step 1.5-amino-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrileand3-amino-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile

3-amino-1H-pyrazole-4-carbonitrile (10 g, 93 mmol) and K₂CO₃ (19.18 g,139 mmol) were taken up in DMF (60 mL). SEM-Cl (19.69 mL, 111 mmol) wasadded at a fast dropwise rate (exotherm) and the resulting mixturestirred at room temperature overnight. Water was added and the productsextracted into EtOAc (×2). The combined organic extracts were washedwith brine, dried over MgSO₄ and concentrated in vacuo. Purification ofthe residue by MPLC (6-50% EtOAc-hexanes) gave5-amino-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile(A) as a pale yellow solid and3-amino-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile(B) as a yellow oily solid.

A—¹H NMR (600 MHz, d6-DMSO): δ 7.54 (s, 1H), 6.77 (s, 2H), 5.21 (s, 2H),3.49 (m, 2H), 0.78 (m, 2H), 0.08 (s, 9H).

B—¹H NMR (600 MHz, d6-DMSO): δ 8.24 (s, 1H), 5.60 (s, 2H), 5.11 (s, 2H),3.47 (m, 2H), 0.79 (m, 2H), 0.07 (s, 9H).

Step 2.5-{[4-(methylsulfonyl)phenyl]amino}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile

5-amino-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile(1.27 g, 5.33 mmol), 4-bromophenyl methyl sulfone (1.253 g, 5.33 mmol),Pd₂ dba₃ (0.244 g, 0.266 mmol), K₂CO₃ (0.810 g, 5.86 mmol) and X-Phos(0.635 g, 1.332 mmol) were taken up in tert-amyl alcohol (15 mL). Theflask was evacuated and back-filled with N₂ (×3) and the resultingmixture was stirred at 100° C. overnight. MeOH was added and the solventremoved in vacuo while loading onto silica. Purification of the residueby MPLC (12-100% EtOAc-hexanes) followed by MPLC (6-100% EtOAc-hexanes)gave5-{[4-(methylsulfonyl)phenyl]amino}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrileas a hygroscopic yellow solid.

¹H NMR (600 MHz, d6-DMSO): δ 9.40 (s, 1H), 8.10 (s, 1H), 7.75 (m, 2H),6.98 (m, 2H), 5.38 (s, 2H), 3.46 (m, 2H), 3.09 (s, 3H), 0.74 (m, 2H),0.17 (s, 9H).

Step 3. 3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile

5-{[4-(methylsulfonyl)phenyl]amino}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazole-4-carbonitrile(0.82 g, 2.089 mmol) was stirred in ethanol (40 mL)/2 N HCl (40 mL) at80° C. for 2 hours. Room temperature was attained, saturated NaHCO₃ wasadded and the products extracted into EtOAc (×2). The combined organicextracts were washed with brine, dried over MgSO₄ and concentrated invacuo to give3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile as a paleyellow solid.

LRMS (APCI) calc'd for (C₁₁H₁₁N₄O₂S) [M+H]⁺: 263, Found: 263.

Step 4.1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile

3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile (64 mg,0.244 mmol), copper(II) acetate (66.5 mg, 0.366 mmol),(4-chlorophenyl)boronic acid (76 mg, 0.488 mmol) and pyridine (0.079 mL,0.976 mmol) were stirred in DCM (8 mL) at room temperature overnight. 2N HCl was added and the products extracted into EtOAc (×2). The combinedorganic extracts were washed with brine, dried over MgSO₄ andconcentrated in vacuo. Purification of the residue twice by MPLC (5-60%EtOAc-hexanes) gave1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrileas a white solid.1-(4-chlorophenyl)-5-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrilewas also isolated.

LRMS (APCI) calc'd for (C₁₇H₁₄ClN₄O₂S) [M+H]⁺: 373, 375, Found: 373,375.

Step 5.1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide

1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile(27 mg, 0.072 mmol) as the starting material.

LRMS (APCI) calc'd for (C₁₇H₁₆ClN₄O₃S) [M+H]⁺: 391, 393, Found: 391,393. JAK2 IC₅₀=290 nM.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples.

TABLE 5 JAK2 Example Characterization IC₅₀ # Structure Compound Name[M + H]⁺ (nM) 16

1-[4- (methylsulfonyl) phenyl]-3-{[4- (methylsulfonyl) phenyl]amino}-1H-pyrazole-4- carboxamide Calc'd 435, found 435 48 17

1-(4-tert- butylphenyl)-3-{[4- (methylsulfonyl) phenyl]amino}-1H-pyrazole-4- carboxamide Calc'd 413, found 413 8.1

Example 18

1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide

Step 1.1-(4-acetylphenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile

1-(4-acetylphenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrilewas prepared according to the general procedure in Example 15 Step 4using 3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile(0.24 g, 0.915 mmol) and (4-acetylphenyl)boronic acid (0.300 g, 1.830mmol) as the starting materials.

LRMS (APCI) calc'd for (C₁₉H₁₇N₄O₃S) [M+H]⁺: 381, Found: 381.

Step 2.1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile

1-(4-acetylphenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile(30 mg, 0.079 mmol) and sodium borohydride (5.97 mg, 0.158 mmol) werestirred in dioxane (1 mL) at 60° C. overnight. Additional sodiumborohydride (5.97 mg, 0.158 mmol) was added and stirring at 60° C.continued for 24 hours. Room temperature was attained, water was addedand the products extracted into EtOAc (×2). The combined organicextracts were washed with brine, dried over MgSO₄ and concentrated invacuo. The residue was triturated in DCM to give1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrileas a white solid.

LRMS (APCI) calc'd for (C₁₉H₁₉N₄O₃S) [M+H]⁺: 383, Found: 383.

Step 3.1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide

1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamidewas prepared according to the general procedure in Example 7 Step 3using1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carbonitrile(15 mg, 0.072 mmol) as the starting material.

LRMS (APCI) calc'd for (C₁₉H₂₁N₄O₄S) [M+H]⁺:401, Found: 401. JAK2IC₅₀=20 nM.

Pharmaceutical Composition

As a specific embodiment of this invention, 100 mg of3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamideis formulated with sufficient finely divided lactose to provide a totalamount of 580 to 590 mg to fill a size 0, hard-gelatin capsule.

Biological Assays JAK1 Enzyme Assay

For the JAK1 enzyme assay, reactions (50 uL) contained 5× IVGN buffer(50 mM Hepes, pH 7.5, 10 mM MgCl₂, 0.01% Brij-35, 1 mM EGTA, 0.1 mg/mlBSA), 2 mM DTT, 2.0 μM peptide substrate, 25 μM MgATP, 400 pM JAK1enzyme and subject compound in 5% DMSO. Reactions were incubated for 60min at RT and quenched with 50 uL 2× quench detect buffer (10 mM EDTA,25 mM HEPES, 0.1% TRITON X-100, 4.7 uM Europium-Py20 and 2.1 mg/mLstreptavidin-APC). Incubate 1 hr at RT and read on a Victor V3 set toread Fluorescent Resonance Energy Transfer (Label 1: Lance 615, Label 2:Lance 665, For both: delay=50 us, window time=100 us, cycle=1000 us,flash energy level=103)

Peptide substrate is amino hexanoyl biotin-EQEDEPEGDYFEWLE-NH2 (SEQ. IDNO.: 1); in DMSO.

JAK2 Kinase Activity Inhibition Assay and Determination of IC₅₀

The kinase activity was measured using a modified version of thehomogeneous time-resolved tyrosine kinase assay described in Park et al.Anal. Biochem. 269, 94-104 (1999).

The procedure for determining the potency of a compound to inhibit JAK2kinase comprises the following steps:

-   -   1. prepare 3-fold serial diluted compound/inhibitor solutions in        100% (DMSO) at 20× of the final desired concentrations in a 96        well plate;    -   2. prepare a master reaction mix containing 6.67 mM MgCl₂, 133.3        mM NaCl, 66.7 mM Tris-HCl (pH 7.4), 0.13 mg/ml BSA, 2.67 mM        dithiothreitol, 0.27 recombinant JAK2 and 666.7 nM biotinylated        synthetic peptide substrate (biotin-ahx-EQEDEPEGDYFEWLE-CONH₂)        (SEQ. ID NO.: 1);    -   3. in a black assay plate, add 2.5 μl compound/inhibitor (or        DMSO) and 37.5 μl master reaction mix per well; initiate the        kinase reaction by adding 10 μl of 75 μM MgATP per well, allow        the reactions to proceed for 80 minutes at room temperate; (the        final conditions for the reactions are: 50 nM JAK2 JH1 domain        (Upstate), 2.0 μM substrate, 15 μM MgATP, 5 mM MgCl₂, 100 mM        NaCl, 2 mM DTT, 0.1 mg/ml BSA, 50 mM Tris (pH 7.4) and 5% DMSO);    -   4. stop the kinase reaction with 50 μl of Stop/Detection buffer        containing 10 mM EDTA, 25 mM HEPES, 0.1% TRITON X-100, 0.126        μg/ml Eu-chelate labeled anti-phosphotyrosine antibody PY20        (cat. # AD0067, PerkinElmer) and 45 μg/ml        Streptavidin-allophycocyanin conjugate (cat. # PJ25S, Prozyme);        and    -   5. read HTRF signals on a Victor reader (PerkinElmer) in HTRF        mode after 60 minutes.

IC₅₀ was obtained by fitting the observed relationship betweencompound/inhibitor concentration and HTRF signal with a 4-parameterlogistic equation.

Compounds of the instant invention are potent inhibitors of recombinantpurified JAK2 kinase activity with an IC₅₀ of approximately 8 nM-1 μM.

JAK3 Enzyme Assay

For the JAK3 enzyme assay, reactions (50 uL) contained 5× IVGN buffer(50 mM Hepes, pH 7.5, 10 mM MgCl₂, 0.01% Brij-35, 1 mM EGTA, 0.1 mg/mlBSA), 2 mM DTT, 2.0 μM peptide substrate, 25 μM MgATP, 400 pM JAK3enzyme and subject compound in 5% DMSO. Reactions were incubated for 60min at RT and quenched with 50 uL 2× quench detect buffer (10 mM EDTA,25 mM HEPES, 0.1% TRITON X-100, 4.7 uM Europium-Py20 and 2.1 mg/mLstreptavidin-APC). Incubate 1 hr at RT and read on a Victor V3 set toread Fluorescent Resonance Energy Transfer (Label 1: Lance 615, Label 2:Lance 665, For both: delay=50 us, window time=100 us, cycle=1000 us,flash energy level=103)

Peptide substrate is amino hexanoyl biotin-EQEDEPEGDYFEWLE-NH2 (SEQ. IDNO.: 1); in DMSO.

TYK2 Enzyme Assay

For the TYK2 enzyme assay, reactions (50 uL) contained 5× IVGN buffer(50 mM Hepes, pH 7.5, 10 mM MgCl₂, 0.01% Brij-35, 1 mM EGTA, 0.1 mg/mlBSA), 2 mM DTT, 2.0 μM peptide substrate, 15 μM MgATP, 125 pM enzyme andsubject compound in 5% DMSO. Reactions were incubated for 60 min at RTand quenched with 50 uL 2× quench detect buffer (10 mM EDTA, 25 mMHEPES, 0.1% TRITON X-100, 4.7 uM Europium-Py20 and 2.1 mg/mLstreptavidin-APC). Incubate 1 hr at RT and read on a Victor V3 set toread Fluorescent Resonance Energy Transfer (Label 1: Lance 615, Label 2:Lance 665, For both: delay=50 us, window time=100 us, cycle=1000 us,flash energy level=103)

Peptide substrate is amino hexanoyl biotin-EQEDEPEGDYFEWLE-NH2 (SEQ. IDNO.: 1); in DMSO.

Assay For JAK Family Protein Kinase Activity

Materials: Streptavidin•allophycocyanin conjugate (SA•APC) andEuropium•cryptate (Eu•K) were from Packard Instrument Company. Eu•Kconjugated pY20 was produced as described in Cummings, R. T.; McGovern,H. M.; Zheng, S.; Park, Y. W. and Hermes, J. D. Use Of APhosphotyrosine-Antibody Pair As A General Detection Method InHomogeneous Time Resolved Fluorescence-Application To HumanImmunodeficiency Viral Protease. Analytical Biochemistry 1999, 33,79-93. Homogenous time resolved fluorescence (HTRF) measurements weremade using the Discovery instrument from Packard. T-stim CultureSupplement was from Collaborative Biomedical Research. Recombinant mouseIL2 was from Pharmingen or R & D.JAK family kinase expression: JAK3, TYK2 and JAK2 kinase domains withN-terminal “Flag” affinity tags were expressed in Sf9 cells usingstandard baculovirus methods. The human JAK3 gene and the human TYK2gene can be purchased from Update (now part of Millpore Corporation).Human JAK2 kinase domain was cloned from a MOLT4 cDNA library(Clonetech).Assay for JAK family protein kinase activity: Tyrosine kinase activitywas measured by detection of the tyrosine phosphorylated peptide aminohexanoyl biotin-EQEDEPEGDYFEWLE-NH2 (SEQ. ID NO.: 1); (S, hereafter)detected by time-resolved fluorescence using a europium labeled antibodyto phosphotyrosine (pY20). The JAK3(JH1) catalyzed phosphorylationreactions were carried out in a 30 uL total reaction volume. Thecompound was run at 5% DMSO and preincubated with enzyme buffer (EB).The EB comprised Invitrogen 5× kinase buffer (50 mM Hepes, pH 7.5, 10 mMMgCl₂, 0.01% Brij-35, 1 mM EGTA, 0.1 mg/ml BSA), 2 mM (final) DTT, 2 μM(final) S, and 250 pM (final) JAK3 enzyme. The assay was run at ATPK_(m) (5 μM final) for 40 to 80 minutes. Reactions were run at ambienttemperature and quenched with an equal volume of quench buffer (QB) (10mM EDTA, 25 mM HEPES, 0.1% TRITON X-100) containing 50 μg/mL SA•APCconjugate and 0.75 nM Eu•K conjugated pY20. This mixture was incubatedat ambient temperature for at least 60 minutes and read on an optimizedfluorescent reader at Ex=320 nm and Em₁=665 nm (SA-APC) and Em₂=615 nM(Eu). The data was analyzed by using a standard 4P fit on the ratio ofthe Em results: (EM₁÷EM₂)*10,000.JAK2 384-Well HEL irfl-bla AlphaScreen™ SureFire™ p-STATS Assay:Principle: When JAK2 is activated and dimerized, it phosphorylates STATSwhich translocates to the nucleus and actives the transcription oftarget genes. AlphaScreen™ SureFire™ p-STAT5 assay (Perkin Elmer and TGRBiosciences) uses both biotinylated anti-phospho-STAT5 antibody, whichis captured by Streptavidin-coated Donor beads, and anti-total STAT5antibody, which is captured by Protein A conjugated Acceptor beads. Theirfl-b/a HEL CellSensor™ cell line was created by transducing parentalHEL 92.1.7 cells (ATCC) with the pLenti-bsd/irfl-bla CellSensor™ vector.When both antibodies bind to phospho-STAT5 proteins released from HELirfl-bla cells, the Donor and Acceptor beads are brought into the closeproximity (<=200 nm) and a cascade of chemical reactions is initiated toproduce a greatly amplified signal. Upon laser excitation, aphotosensitizer in the donor bead converts ambient oxygen to a moreexcited singlet state. The singlet state oxygen molecules diffuse acrossto react with a chemiluminescer in the acceptor bead that furtheractivates fluorophores contained within the same bead. The fluorophoressubsequently emit light at 520-620 nm. The emitted light intensity isdirectly proportional to the amount of phospho-STAT5 proteins releasedfrom HEL irfl-bla cells.Growth Medium: RPMI Medium 1640 (Invitrogen) with 10% dialyzed FBS(Invitrogen), 1 μg/ml blasticidin, 0.1 mM NEAA, 1 mM sodium pyruvate and1% Pen-Strep.Method: On day 1, split HEL irfl-bla cells at density of 500,000cells/ml. Incubate cells in a tissue culture flask at 37° C., 5% CO₂overnight. On day 2, harvest cells and wash the once with HBSS(Invitrogen) containing 0.5% dialyzed FBS. Next, seed cells at a densityof 100,000 cells/well in 8 ul of HBSS w/0.5% dialyzed FBS in 384-wellmicroliter plates. Temporarily put these cell plates in a 37° C., 5% CO₂incubator. To prepare a compound plate, prepare serially dilutedcompounds in DMSO at a 500× stock concentration. Transfer 2 uL of theserially diluted compounds from the compound plate to an intermediatedilution plate containing 198 uL of HBSS w/ 0.5% dialyzed FBS. Next,transfer 2 uL of intermediately diluted compounds to each well of thecell plate to get 1:500 final dilution of each test compound andcontrols. Incubate the cell plates at 37° C., 5% CO₂ for 1 hr. Add 2.5ul/well of 5× lysis buffer from the kit to cell plates. Gently agitatethe plates for 5-10 min.

Make detection reagent mixture A by adding together 800 uL reactionbuffer, 20 uL acceptor beads, and 200 uL activation buffer. Add 15uL/well of detection mixture A to the cell plates and gently agitate theplates for 1-2 min. Seal the plates with an adhesive cover and incubateat room temperature for 2 hr, avoiding exposure to light. Make detectionmixture B by adding together 400 uL dilution buffer and 20 uL donorbeads. Add 6 uL/well of mixture B to the cell plates and gently agitatethe plates for 1-2 min. Seal the plates with an adhesive cover andincubate at room temperature for 2 hr, avoiding exposure to light. Readthe plates on an AlphaScreen-capable plate reader.

Cellular proliferation assays: CTLL-2 cells (ATCC) were maintained in 6%T-stim Culture Supplement (source of IL2) in RPMI-1640 supplemented with10% fetal bovine serum, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol,1.4 mM L-glutamine, 10 mM HEPES, 1 mg/ml dextrose, 0.04 mM essentialamino acids, 0.02 mM nonessential amino acids, penicillin andstreptomycin (H10). The day before use in the proliferation assay, cellswere washed and resuspended in 0.2% Tstim at a cell concentration of5×10⁵/ml. The next day, cells were washed and plated at 0.2-1×10⁵cells/well in a 96 well tissue culture plate (CoStar). 0.05 ng/ml mouserecombinant IL2 (Pharmingen), with or without a test compound, or 20ng/ml PMA (Sigma) and 1 μCi/well [³H]-thymidine were added. Afterovernight culture, cells were harvested with a glass fiber Filtermat(Wallac) and a Tomtek cell harvester. Tritium incorporation was measuredby liquid scintillation counting on a Topcount scintillation counter(Packard).

Compounds of the instant invention are inhibitors of recombinantpurified JAK3 kinase activity with an IC₅₀ of approximately >3000 nM.

While a number of embodiments of this invention have been described, itis apparent that the basic examples may be altered to provide otherembodiments, encompassed by the present invention. Therefore, it will beappreciated that the scope of this invention is to be defined by theappended claims rather than by the specific embodiments, which have beenrepresented by way of example.

1. A compound of the following formula:

wherein R¹ is aryl, which is optionally substituted with one to threesubstituents independently selected from the group consisting of halo,hydroxyl, cyano, SO₂R³, C₁₋₆ alkyl and heterocyclyl; wherein said alkylgroup is optionally substituted with one to three halo, hydroxyl orcyano, and said heterocyclyl group is optionally substituted on eitherthe carbon or heteroatom with one to three halo, hydroxyl or oxo; R² isaryl or heteroaryl, wherein said aryl and heteroaryl groups areoptionally substituted on either the carbon or heteroatom with one tothree substituents selected from the group consisting of halo, cyano,C₁₋₆ alkyl, heterocyclyl, (C═O)heterocyclyl, (C═O)R³, (C═O)OR³,(C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴, NR³SO₂R⁴ and SO₂(heterocyclyl); whereinsaid alkyl group is optionally substituted with one to three halo,hydroxyl, O(C═O)NR³R⁴, heteroaryl, heterocyclyl or NR³R⁴; saidheterocyclyl groups are optionally substituted on either the carbon orheteroatom with one to two groups independently selected from the groupconsisting of C₁₋₃ alkyl, hydroxyl and oxo; and said heteroaryl groupsare optionally substituted on either the carbon or heteroatom with C₁₋₃alkyl; R³ is hydrogen or C₁₋₆ alkyl; R⁴ is hydrogen or C₁₋₆ alkyl; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1wherein R¹ is aryl, wherein said aryl group is substituted with one tothree substituents independently selected from the group consisting ofhalo and C₁₋₆ alkyl, wherein said alkyl group is optionally substitutedwith hydroxyl; or a pharmaceutically acceptable salt thereof.
 3. Thecompound of claim 2 wherein R¹ is phenyl, wherein said phenyl group issubstituted with one to three halo; or a pharmaceutically acceptablesalt thereof.
 4. The compound of claim 1 wherein R² is aryl orheteroaryl, wherein said aryl and heteroaryl groups are optionallysubstituted on either the carbon or heteroatom with one to threesubstituents selected from the group consisting of C₁₋₆ alkyl,(C═O)heterocyclyl, (C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴ and SO₂(heterocyclyl);wherein said alkyl group is optionally substituted with hydroxyl,heterocyclyl or NR³R⁴; said heterocyclyl groups are optionallysubstituted on either the carbon or heteroatom with one to two groupsindependently selected from the group consisting of C₁₋₃ alkyl and oxo;or a pharmaceutically acceptable salt thereof.
 5. The compound of claim4 wherein R² is aryl, wherein said aryl group is optionally substitutedwith one to three substituents selected from the group consisting ofC₁₋₆ alkyl, (C═O)heterocyclyl, (C═O)NR³R⁴, SO₂R³, SO₂NR³R⁴ andSO₂(heterocyclyl); wherein said alkyl group is optionally substitutedwith hydroxyl, heterocyclyl or NR³R⁴; said heterocyclyl groups areoptionally substituted on either the carbon or heteroatom with one totwo groups independently selected from the group consisting of C₁₋₃alkyl and oxo; or a pharmaceutically acceptable salt thereof.
 6. Thecompound of claim 5 wherein R² is phenyl, wherein said phenyl group isoptionally substituted with one to three substituents selected from thegroup consisting of C₁₋₆ alkyl, (C═O)heterocyclyl, (C═O)NR³R⁴, SO₂R³,SO₂NR³R⁴ and SO₂(heterocyclyl); wherein said alkyl group is optionallysubstituted with hydroxyl or heterocyclyl; said heterocyclyl groups areoptionally substituted on either the carbon or heteroatom with one totwo groups independently selected from the group consisting of C₁₋₃alkyl, and oxo; or a pharmaceutically acceptable salt thereof.
 7. Thecompound of claim 1 selected from3-{[4-(methylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(methylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-{[2-(hydroxymethyl)pyridin-4-yl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;3-{[4-(morpholin-4-ylcarbonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;3-{[4-(morpholin-4-ylsulfonyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(dimethylamino)sulfonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(dimethylamino)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-anilino-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(1,1-dioxidothiomorpholin-4-yl)carbonyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-{[4-(morpholin-4-ylmethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-({4-[(dimethylamino)methyl]phenyl}amino)-1-phenyl-1H-pyrazole-4-carboxamide;3-{[4-(1-hydroxy-1-methylethyl)phenyl]amino}-1-phenyl-1H-pyrazole-4-carboxamide;1-(4-chlorophenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;1-[4-(methylsulfonyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;1-(4-tert-butylphenyl)-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;1-[4-(1-hydroxyethyl)phenyl]-3-{[4-(methylsulfonyl)phenyl]amino}-1H-pyrazole-4-carboxamide;or a pharmaceutically acceptable salt thereof.
 8. A pharmaceuticalcomposition comprising a pharmaceutically effective amount of thecompound according to claim 1, and a pharmaceutically acceptablecarrier.
 9. A method of treating myeloproliferative disorders or cancerin a mammal in need thereof with a compound according to claim 1.